Gold 9723725 The lignin degradative system (LDS) of the white-rot basidiomycetous fungus Phanerochaete chrysosporium degrades the plant cell wall polymer, lignin, and a wide range of aromatic pollutants. The major extracellular components of this nonspecific oxidative system are two heme-containing peroxidases, manganese peroxidase (MnP) and lignin peroxidase (LiP). The LDS is expressed during secondary metabolic growth, the onset of which is triggered by the depletion of nutrient nitrogen. MnP expression also is regulated at the level of gene transcription by Mn ion, by heat shock, and by oxidative stress. The primary goal of this project is to elucidate these specific mechanisms of mnp gene transcription at the molecular level. New mnp promoter-gene reporter constructs will be used to examine the regulation of individual mnp genes by nitrogen, Mn, and heat shock. The cis-acting promoter sequences involved in mnp regulation using site-directed mutagenesis of putative metal response elements, promoter deletion analysis, and synthetic promoter elements will be identified. The putative Mn-responsive transcription factor will be identified and purified, and genes encoding the Mn-responsive and heat shock transcription factors will be cloned. Finally, the post-transcriptional requirements for MnP synthesis will be examined further. Lignin is the second most abundant natural polymer, constituting 15-30% of woody plant cell walls, and forming a matrix that surrounds the cellulose in wood. Thus, the degradation of lignin is a significant step in the global carbon cycle. Furthermore, lignin presents a major obstacle to the industrial utilization of cellulose. White-rot fungi are the only organisms which can completely degrade lignin to carbon dioxide and water. The best studied of these fungi, Phanerochaete chrysosporium, efficiently degrades lignin and a wide range of environmental pollutants. P. chrysosporium and other lignin-degrading fungi secrete the enzyme manganese peroxidase (Mn P). This enzyme oxidizes manganese ion (Mn) which, in turn, depolymerizes lignin. The production of MnP is regulated at the level of the gene by the depletion of nutrient nitrogen, by the availability of Mn, and by temperature and chemical stresses. The goal of this research is to understand, at the molecular level, the mechanisms by which each of these factors control MnP production. This is the first example of Mn regulation of gene expression to be studied at the molecular level.
